Elucidating the glycan-binding specificity and structure of Cucumis melo agglutinin, a new R-type lectin

Plant lectins have garnered attention for their roles as laboratory probes and potential therapeutics. Here, we report the discovery and characterization of Cucumis melo agglutinin (CMA1), a new R-type lectin from melon. Our findings reveal CMA1’s unique glycan-binding profile, mechanistically explained by its 3D structure, augmenting our understanding of R-type lectins. We expressed CMA1 recombinantly and assessed its binding specificity using multiple glycan arrays, covering 1,046 unique sequences. This resulted in a complex binding profile, strongly preferring C2-substituted, beta-linked galactose (both GalNAc and Fuca1-2Gal), which we contrasted with the established R-type lectin Ricinus communis agglutinin 1 (RCA1). We also report binding of specific glycosaminoglycan subtypes and a general enhancement of binding by sulfation. Further validation using agglutination, thermal shift assays, and surface plasmon resonance confirmed and quantified this binding specificity in solution. Finally, we solved the high-resolution structure of the CMA1 N-terminal domain using X-ray crystallography, supporting our functional findings at the molecular level. Our study provides a comprehensive understanding of CMA1, laying the groundwork for further exploration of its biological and therapeutic potential.


Method of preparation:
Please see the Experimental section in the main text.
Included for comparison is the biotinylated plant lectin Ricinus Communis Agglutinin I (RCA1) from Vector Laboratories.

Sample modifications
Not relevant.

Assay protocol
Microarray analyses were performed essentially as described (Liu et al., Methods Mol. Biol. 2012), for modifications of the protocols please see "Glycan array experiments" under Experimental section in the main text.

Glycan description for defined glycans
A screening microarray of sequence-defined lipid-linked glycan probes (neoglycolipids, NGLs For full description on the definition of lipid moieties of the glycan probes please see https://glycosciences.med.ic.ac.uk/docs/lipids.pdf

Description of surface
Nitrocellulose-coated glass microarray slides.

Custom preparation of surface
Not relevant.

Dispensing mechanism
Non-contact liquid delivery with four dispensing tips.

Glycan deposition
Approximately 0.33 nl was printed per spot.
Lipid-linked glycan probes were printed at 2 and 5 fmol per spot, and polysaccharides at 0.03 and 0.1 ng per spot, all in duplicate.

Printing conditions
The printing solutions were all aqueous based.Printing was performed at ambient temperature and relative humidity of 58%.
The 'liposome' printing solutions contained 100 pmol/µl of DHPC and cholesterol (both from SIGMA) as lipid carriers in addition to the lipid-linked glycan probes.The concentrations of the lipid-linked glycan probes were 5 and 15 pmol/µl for the 2 and 5 fmol per spot levels, respectively.
The printing solutions also contained Cy3 NHS ester (GE Healthcare) at 20 ng/ml (26 fmol/µl) as a marker to monitor the printing process.

Array layout
Each array slide contained 16-pad subarrays.Each pad was set up for printing 64 probes maximum, each at 2 levels in duplicate (four spots for one probe in a row); up to 256 spots (16x16) in total in each pad.
The 866 lipid-linked probes in the screening arrays were printed on multiple subarrays for parallel binding analyses.

Glycan identification and quality control
The quality control of the screening microarrays of sequence-defined glycan probes was carried out with a collection of biotinylated plant lectins including Concanavalin A (ConA), Aleuria aurantia lectin (AAL) and wheat germ agglutinin (WGA) (all from Vector Laboratories).The immobilization of the GAG oligosaccharide probes was evaluated with human FGF2, anti-CS and anti-KS antibodies (Wu et al. Mol Cell Proteomics. 2019).These data will be described elsewhere and are available upon request.
The glycan probes that may have inaccurate immobilization amounts, as indicated by our quality control experiments, are shaded in grey in Supplementary Table 4. Laser power used for scanning the arrays to achieve maximum signal without spot saturation: 50% for CMA1; 15% for RCA1.

Detector and Data Processing
Image analysis software GenePix® Pro 7 (Molecular Devices)

Data processing
The gpr files were entered into an in-house microarray database using software (designed by Mark Stoll, http://www.beilsteininstitut.de/en/publications/proceedings/glyco-2009)for data processing.No particular normalization method or statistical analysis was used for the results of the screening arrays.
Data were transformed into z-scores by subtracting the mean value across the array and dividing the results by the standard deviation.

Data presentation
The microarray binding results are in Figure 2, and Supplementary Table 2.

Data interpretation
No software or algorithms were used to interpret processed data.

Conclusions
At the assay condition used, the recombinant CMA1 lectin showed binding showed a broad range of binding activities and bound four types of glycan sequences: galactose (Gal)-terminating, GalNAc-terminating, fucose (Fuc)-terminating and chondroitin sulphate (CS) related sequences.Further details regarding the specificity of the lectin are elaborated upon in the main text.